果蝇来源的GPCR Methuselah G蛋白偶联信号转导通路研究  

Characterization of G Protein-coupled Signal Transduction of the Drosophila GPCR Methuselah

作　　者：张静[1,2] 张儒[1] 叶晨立[1,2] 谢欣[1,2] 

机构地区：[1]上海市信号转导与疾病研究重点实验室同济大学生命科学与技术学院,上海200092 [2]中国科学院上海药物研究所国家新药筛选中心,上海201203

出　　处：《中国细胞生物学学报》2011年第8期847-854,共8页Chinese Journal of Cell Biology

基　　金：科技部科研项目(No.2008DFB30150);上海市科委科研(No.08431910100;No.09DZ2260100;No.2010CB944901;No.2011CB965104)资助项目~~

摘　　要：Methuselah(MTH)是果蝇来源的GPCR中的一员,它的突变可延长果蝇平均寿命并提高果蝇对外界胁迫因素的耐受性。但目前对MTH在细胞水平的信号转导研究鲜有报道。该研究用稳定表达MTH的HEK293细胞株,对与该受体偶联的G蛋白选择性做了研究。首先,用免疫荧光染色、Western blot及钙流实验验证了MTH在HEK293/Myc-MTH细胞表面能稳定表达,且具有正常生物学活性;MTH受体被其配体N-stunted活化后所引起细胞内钙的上升不能被PTX预处理抑制,提示活化的MTH可能通过与Gq/11而非Gi/o蛋白相偶联;进一步研究发现,MTH激活后不显著改变细胞中的cAMP水平,表明MTH不与Gs和Gi/o相偶联;MTH被激活后可引起ERK磷酸化。这些结果提示:MTH可能是Gq/11蛋白的偶联受体,为进一步研究MTH的下游信号转导和生物学功能奠定了基础。Drosophila Methuselah （MTH） is a member of the G protein-coupled receptor （GPCR） super family. Mutation of MTH receptor leads to extended life span and resistance to various forms of stress in fruit fly. However, the signal transduction of MTH at cellular level has rarely been studied. Using HEK293 cell line which stably expresses MTH, we investigated its G protein-coupling preference. Firstly, we confirmed the expression and biological activity of MTH in HEK293 cells by using immunofluorescent staining, Western blot analysis and calcium mobilization assay. N-stunted, the endogenous ligand of MTH, induced calcium mobilization in cells expressing MTH. The calcium signal was insensitive to PTX pretreatment, indicating MTH is probably coupling to Gq/11 rather than Gi/o pathway. Then we discovered that activation of MTH did not induce any change in the intracellular cAMP level in HEK293 cells, suggesting that MTH is not coulpled to Gs or Gi/o. Finally, we showed that activation of MTH could induce MAP kinase ERK1/2 phosphorylation. Our results revealed major signal transduction pathways of MTH which might facilitate further research of this receptor and help us understanding its biological functions.

关 键 词：G蛋白偶联受体 Methuselah受体 G蛋白 信号转导 

分 类 号：Q51[生物学—生物化学]